Triacylglycerols (TGs) are quantitatively the most important storage form of energy for eukaryotic cells, and TG synthesis plays an important role in metabolism in the intestine, liver, mammary gland, and adipose tissue. TGs are synthesized from fatty acyl CoA and diacylglycerol, which can be derived from the glycerol-3-phospate pathway or, in intestinal enterocytes, from monoacylglycerol. The enzyme acyl CoA:diacylglycerol acyltransferase (DGAT) has long been recognized as a key enzyme in TG synthesis and has been thought to catalyze the terminal and only committed step in the pathway. We recently cloned a DGAT gene and now have inactivated this gene in mice. Surprisingly, the DGAT knockout mice are viable and have normal plasma TGs and significant amounts of TGs in adipose tissue, revealing that an additional mechanism for TG synthesis exists. Importantly, another mechanism must participate in TG synthesis for incorporation into lipoproteins. Additionally, although they have adipose tissue, DGAT-deficient mice are leaner than their chow-fed littermates and are resistant to developing obesity when they are fed a high-fat diet. This finding has identified DGAT inhibition as a possible strategy for treating obesity. This revised proposal has three major aims. Specific Aim 1 is to determine the expression pattern and regulation of DGAT. We will determine the tissue expression pattern of DGAT mRNA and protein by techniques such as immunoblotting and immunohistochemistry, and we will examine the regulation of DGAT mRNA and protein expression levels in response to metabolic perturbations. Specific Aim 2 is to investigate further the in vivo functions of DGAT. Specifically, we will determine: 1) the mechanism for the leanness and obesity resistance in DGAT knockout mice, 2) whether DGAT deficiency protects against obesity and insulin resistance syndromes caused by genetic mutations, and 3) the effects of DGAT deficiency on the synthesis and secretion of intestinal chylomicrons and hepatic very low density lipoproteins. Specific Aim 3 is to identify and clone a second DGAT enzyme or other enzymes that catalyze TG synthesis. We will analyze the biochemistry of alternative pathways of TG synthesis in DGAT- deficient tissues such as the small intestine, liver, and adipose tissue, and in Dgat-/- fibroblasts. Additionally, we will use a retrovirus-based expression cloning strategy in Dgat-/-, fibroblasts to identify and clone genes that augment TG synthesis. The proposed studies should yield important new information concerning triacylglycerol synthesis in mammals.